Mucosal and cutaneous epithelial surfaces act as a barrier to microbial infection. For decades, this barrier was thought to represent only a physical impediment to microbial colonization, dependent upon innate cellular and adaptive immunity to recognize and eliminate foreign antigens. Recently, work from our laboratory and others have identified additional components of the mucosal defense system used to resist infection and respond to injury. These "antimicrobial peptides" consist of diverse gene families and protein structures and can be found in plants, insects, amphibians, and mammals. At least three families of antimicrobial peptides are produced in human mucosal epithelium: alpha-defensins, beta-defensins, and the more recently discovered cathelicidins. Cathelicidins represent a unique family of mucosal antimicrobial peptides that do not undergo the extensive posttranslational modifications necessary for production of active defensins. This proposal seeks to exploit this fundamental advantage of the cathelicidins and use a bacterial genetic approach to overexpress these peptides and understand their mechanism(s) of action. Specifically, this investigation proposes to: (1) determine the antimicrobial activity of cathelicidins against important human mucosal pathogens and prevalent commensal microflora; (2) investigate mechanisms of microbial sensitivity and resistance to cathelicidins by bacterial mutagenesis and cloning strategies. Clone and characterize bacterial genes conferring resistance or susceptibility to cathelicidins; and (3) express a functional mammalian cathelicidin in a prokaryotic background as a first step toward development of a novel antimicrobial peptide delivery system for therapy of human mucosal infections. This exploratory project, bringing together investigators with complementary expertise, may enhance our understanding of the role of cathelicidins in mucosal immunity, and potentially establish new therapeutic targets and strategies to combat human mucosal pathogens.